A hallmark of the HLA complex in man is polymorphism (i). Like class I and II loci the class III fourth component of complement loci, i.e. C4A and C4B, are highly polymorphic with more than 35 alleles. Genetic studies have mapped susceptibility to a number of diseases, in particular immune complex (ic) disease, to this region of the HLA. The long term objective of this proposal is to understand the functional importance of the genetic polymorphism and how it might be related to the immune response and disease susceptibility. This objective has been organized into three specific aims: (1) Determine the structural basis and functional significance of C4 polymorphism; (2) Determine the affect of genetic variation of the C4 isotypes on the human immune response; (3) Develop genetic models for the analysis of the role of C4 protein in the immune response in vivo. The first aim proposes to test the hypothesis that both the isotypic and allotypic residues affect covalent binding to the ic. Human C4A and C4B coding sequences will be mutated using site-directed mutagenesis. Understanding the affect these residues have on binding is important since the efficiency of binding directly determines the extent of activation of the pathway. The second aim of this proposal is to test the hypothesis that non- response to hepatitis B vaccine is due to homozygous C4A null alleles. This will be tested by extending the previous study to include individuals that are homozygous for the C4A null allele but on HLA haplotypes other than B8 DR3. The finding that C4 isotype affects the immune response to certain vaccines would be of significant importance. The third aim proposes to develop a C4 deficient strain of mouse. This strain will be used in this proposal for: (1) characterization of the role of complement in the immune response; (2) genetic manipulation such as insertion of transgenes of either human C4A or C4B; (3) direct comparison of the immune response by C4A and C4B transgenic mice; and (4) developing a strategy for a replacement therapy in humans. In summary, the combined approach of (1) biochemical studies on the importance of the structural variation of C4; (2) vaccination of humans homozygous for either C4A or C4B null alleles with two different antigens; and (3) construction of C4 deficient and C4A/C4B transgenic mice for biological studies on both the role of C4 in the immune response and the affect of polymorphism on its role; will answer important questions that will lead to future therapy against human disease.